Two P450 cytochromes from beef adrenal mitochondria which catalyze specifically side chain cleavage of cholesterol (P450scc) and steroid 11 beta-hydroxylation have been separated and purified to better than 70 percent of purity. Amino acid analysis and heme peptide formation (with K. Dus, St. Louis) indicate close similarity to bacterial P450 cytochromes. Spectroscopic analysis of P450scc in rat adrenal mitochondria indicates two forms of the cytochrome and 3 steroid binding sites. ACTH action in vivo effects the combination of cholesterol with one binding site which is characterized by pH and temperature sensitivity. Corticosterone formation in isolated rat adrenal cells was decreased by protein synthesis inhibitors in a manner distinct from protein synthesis. These inhibitors were significantly more potent in the inhibition of ACTH stimulated steroidogenesis than in inhibition of the cAMP stimulated process. These observations are consistent with the hypothesis that ACTH action requires a labile protein which is stabilized by a cAMP-mediated process. Steroidogenesis was also diminished by inhibitors of microtubule and microfilement formation. These inhibitors, like protein synthesis inhibitors, decreased the combination of cholesterol with adrenal mitochondrial P450scc in vivo. Experiments with pig corpus luteum indicated that movement of cholesterol to mitochondrial P450scc was also hormonally regulated and was the earliest measurable biochemical change in the luteolytic (or PGF 2alpha induced) decrease in progesterone production.